Résumé :
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The core objective of this study is to help elucidate the functional pathogenesis of nemaline myopathy and related disorders caused by mutations in the nebulin gene (NEB). Nebulin is a giant actin-binding protein which is located in the I-band of the sarcomeres of skeletal muscle. It is a very large protein (600-900 kDa) and it is believed that nebulin acts as a thin filament "ruler" and regulates thin filament length during sarcomere assembly. Apart from thin filament regulation, the structural and functional roles of nebulin expand to maintaining intermyofibrillar activity, setting physiological Z-disk widths and calcium-handling in the skeletal muscles, thus having a specific role in the regulation of muscle contraction. Nebulin has binding sites for an array of diverse proteins. The N-terminus of nebulin interacts with tropomodulin at the pointed end of the thin filament and the C-terminus binds myopalladin, titin and CapZ in the Z disc, while nebulin domains close to the Z disc bind desmin. Many functions of nebulin, such as a role in cell signalling, are currently unknown. We have initiated functional studies of nebulin to explore the pathogenetic pathways that NEB mutations lead to, and to find out the co-relation between these pathways and the structural abnormalities seen in the muscle fibres and in the clinical phenotypes. The exons selected for the pathogenetic studies include the Ashkenazi founder mutation (exon 55 deletion) and Finnish founder mutations (in exons 122, 151) along with other mutations (in exons 54, 78). After successful cloning of these exons, protein production was optimised and F-actin binding assays were performed. There seems to be some differences in binding affinities for actin between wild type and mutated/deleted nebulin fragments. To better understand the physiological roles of exons 143/144 in producing different isoforms, and to find the interacting partners of these nebulin domains, we screened the human skeletal Matchmaker cDNA library by using the yeast two-hybrid system with exons 143/144 as baits. After the initial interaction studies, the results were further verified by using different bait and prey combinations together with positive and negative controls. Our studies have revealed interesting candidate binding partners for the exons in question. The findings using yeast two-hybrid system have also been confirmed using co-immunofluorescence. The study is ongoing.
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